DNA tecnology Flashcards
What is the different between a Southern and Northern Blot?
A southern blot is use ssDNA and RNA probes to detect specific DNA sequences.
Northern blot uses probes to look for RNA sequences.
What is in situ hybridization used for?
In situ hybridisation is a technique used for physicaly finding and detecting specific DNA and RNA sequences in cells.
What are the differences between blots and arrays?
Blots are used to find DNA or RNA sequences (Identification of proteins and nucleic acids), and samples are immobilised to solid support, single probe in introduced in liquid phase.
Arrays: Used for DNA expression analysis. Many probes are immobilised to solid support, samples are introduces in liquid phase.
A PCR cycle includes 3 phases, what are these?
A PCR includes: DNA polymerase, Primer (Forward + reverse) and dNTP.
The 3 phases are Denaturation, annealing and extension.
(1) denaturation of the template into single strands; (2) annealing of primers to each original strand for new strand synthesis; and (3) extension of the new DNA strands from the primers.
What does Sanger sequencing use to stop primer extension reactions?
Di-deoxynucleotides
What are the main differences between Sanger and High-throughput sequencing?
Sanger sequencing requires pre exisiting knowledge about the target, it reads 600-800 nts, is expensive and slow. Chain termination.
HTS does not require any pre knowledge about the target, Reads 100-100 000 nts, is cheap and fast.
I digest 2 human DNA samples, one with HindIII (A ↓ AGCTT) and one with Dpn1 (GA↓TC),
which gives more fragments and what is the geometry of the cut ends?
Dpn1 will give more fragments because its recognition site is smaller and will occur more
frequently in the genome. Dpn1 produces a blunt end, HindIII will be sticky (cohesive).
In what ways can I “pimp” a PCR?
Use specialized DNA polymerase, chemically modified primers, PCR additives to improve
performance, extended primer sequences.
What is a genomic library?
a collection of DNA fragments contained within self-replicating vectors that represent the entire genome of the individual from which the DNA was made.
I have a library of DNA clones where the inserts are (on average) 20Kb long. What sort of vector
have I most likely used as a host?
Lambda phage
What produces cDNA and what template does it use?
Reverse Transcriptase uses mRNA as a template to produce cDNA
